人肝再生增强因子真核表达及其对顺铂诱导肝癌细胞凋亡的影响

Eukaryotic expression of human augmenter of liver regeneration and its effects on the apoptosis of QGY cells induced by cisplatin

摘要:

目的：构建人肝再生增强因子（human augmenter of liver regeneration，hALR）15kD亚型的真核表达系统，并探索ALR对顺铂（Cisplatin，DDP）诱导的人肝癌细胞株QGY凋亡的影响。方法：用聚合酶链反应（polymerase chain reaction，PCR）方法和基因重组技术，从重组质粒pPIC9K-rhALR中扩增出编码rhALR基因片段，将其克隆入pPICZαA质粒中构建重组质粒pPICZα－A-rhALR。重组质粒SacⅠ酶切线性化后电转入酵母GS115中，并在1%的甲醇诱导下表达。表达上清蛋白经Western blot（ALR多克隆抗体和His-tag标签抗体）鉴定和镍柱亲和层析纯化。MTS试剂检测rhALR体外对人肝癌细胞（HepG2、QGY）的促增殖活性，及流式细胞仪检测rhALR在顺铂诱导QGY细胞凋亡中的抗凋亡作用。结果：PCR、双酶切、DNA测序均鉴定重组质粒构建与预期一致。分泌表达的rhALR约占上清总蛋白的70%，目的分子量约17 kD，Western blot均可见单一条带。纯化后的rhALR对HepG2和QGY的体外促增殖作用呈浓度依赖性增强（HepG2组：F=246.729，P=0.000；QGY组：F=246.004，P=0.000），且在QGY细胞顺铂诱导凋亡时也发挥着浓度梯度式抗凋亡作用（F=101.061，P=0.000）。结论：成功构建高效分泌表达rhALR的pPICZα－A-rhALR GS115真核表达系统；体外实验证实rhALR对顺铂诱导人肝癌细胞有呈浓度依赖性的抗凋亡作用。

Abstract:

Objective：To construct pichia pastoris GS115 expression system of 15 kD recombinant human augmenter of liver regener－ation（rhALR），and to explore its effects on human hepatoma cell line QGY treated with cisplatin（DDP）. Methods：With polymerase chain reaction（PCR），and gene recombination techniques，cDNA of rhALR was obtained from recombinant plasmid pPIC9K-rhALR and inserted into plasmid pPICZαA. The recombinant plasmid pPICZαA-rhALR demonstrated by sequencing was linearized by di－gestion with SacⅠ，and transformed into GS115 with electroporation. The rhALR expression was secreted by GS115 under the induction of 10 mL/L methanol，and purified through Nickel column affinity chromatography after it was analyzed by Western blot（ALR poly－clonal antibody and His-tag mouse monoclonal antibody）. The effects of rhALR on in vitro proliferation of QGY and HepG2 cells，as well as its effects on reducing cellular proliferation inhibiton rate induced by DDP，were evaluated by MTS reagent. The anti-apoptosis effects of rhALR on QGY cells apoptosis induced by DDP were detected by flow cytometry. Results：The recombinant plasmid pPICZα－A-rhALR was identified by PCR，restriction enzyme reaction methods and direct sequencing respectively. RhALR as a secretory pro－tein was successfully expressed by GS115；with molecular weight about 17 kD，it accounts for 70% of the total protein in the super－natant from pPICZαA-rhALR GS115. The results of Western blot all showed the specific single band. The high qualitative rhALR pu－rified through Nickel column affinity chromatography could stimulate in vitro proliferation of human hepatoma cell lines（HepG2 and QGY） in a dose-dependent manner（F=246.729，P=0.000；F=246.004，P=0.000），reduce cellular proliferation in－hibition rate and play anti-apoptosis effect also in a dose-de－pendent manner when QGY cells treated with DDP（F=101.061，P=0.000）. Conclusion：The rhALR as a secretory protein is expressed in GS115 efficiently and purified through Nickel column affinity chromatography successfully. The rhALR exerts anti-apoptosis effects on QGY cells treated with DDP in a dose-dependent manner in vitro.